AB0007 DYNAMIC INTERPLAY OF FUNCTIONALLY DISCORDANT CD4+HLA-DR+ SUBSETS WITH A COMMON PATHOLOGICAL ONTOGENIC ORIGIN IN HUMAN ARTHRITIS
Authors: J. Y. Leong, P. Kumar, A. J. M. Lim, S. H. Tay, S. Hazirah, J. G. Yeo, C. Chua, P. Chen, A. M. Lajam, A. Consolaro, M. Gattorno, C. T. Ng, T. Arkachaisri, A. Martini, S. Albani
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> We have previously described the presence of pathogenic-like lymphocyte (CPLs; CD4<sup>+</sup>HLA-DR<sup>+</sup>) subset within the circulation, that is phenotypically similar to their synovial counterparts. CPLs are inflammatory T effectors that correlate with disease activity and possess a TCR repertoire with strong synovial overlap. We also discovered a similar inflammation-associated T-regulatory (iaTreg; CD4<sup>+</sup>HLA-DR<sup>+</sup>) circulatory subset, that is activated, suppressive, poised to migrate to inflamed site and sharing TCR between the peripheral and synovial compartments. iaTreg and CPL were found to be phenotypcally quite similar. Their striking similarity, inspired the hypotheses of a common ontogeny driven by the synovial micro-environment, and whether their functionally discordant roles, pointed to an undetermined dynamic interplay. <h3>Objectives</h3> In this study, we seek to establish if a common disease ontogeny exists between synovial CPLs/iaTregs in human arthritis and why CPLs continue to perpetuate synovial inflammation. <h3>Methods</h3> We performed mass cytometry analysis of PBMCs from RA patients and healthy adults. CD4 subsets from active JIA PBMCs, paired JIA SFMCs and healthy paediatric controls were sorted for bulk RNA sequencing. CpG profiling of FoxP3 TSDR site was performed for synovial iaTregs. Treg suppression assays were performed in iaTregs for healthy paediatric controls, paired JIA PBMCs and SFMCs. <h3>Results</h3> Mass cytometry analysis of RA patients revealed memory induction of CPLs and iaTreg as compared with healthy adults. Importantly, CPLs/iaTregs from RA patients demonstrated reactivity to the dnaJP1 peptide, which is a pro-inflammatory epitope in RA patients, thus underscoring disease relevance. Circulatory JIA CPLs/iaTreg subsets displayed transcriptomic convergence, exhibiting (a) common pathway dysregulations, (b) restriction in TCR repertoire and (c) common transcription factor drivers, suggesting a common pathogenic ontogeny. This phenomenon is further reinforced across the spatial/disease continuum, with synovium CPLs/iaTregs presenting common dysregulated pathways including IFNg signalling. In particular, TCR clonatype sharing in CPLs/iaTregs is higher in the synovium, suggesting a common antigenic origin. To understand the dynamic interplay between CPLs and iaTregs, we assess the regulatory capacity of iaTregs. Epigenetic profiling of CpG sites on the FoxP3 TSDR promoter, revealed typical Treg demethylation status within iaTregs, indicating a stable lineage. Suppression assays reveal that iaTreg rather than non-iaTregs were poised to suppress disease T effectors from JIA PBMC/SFMC, with higher rates of suppression. This is in contrast with healthy controls, where iaTregs were inferior in suppression, mirroring their disease ontogeny. Despite superior suppression from iaTreg in SFMC T effectors, synovial CPLs were resistant to suppression from iaTreg as compared with synovial Non-CPLs. Destabilisation assays in iaTregs, reveal a drop in FoxP3 levels in the presence of IFNg, suggesting a role for IFNg in CPL evasion of suppression. <h3>Conclusion</h3> Our study highlights the disease relevance of CPLs/iaTregs in human arthritis, whilst revealing evidence of a common pathological ontogeny for CPLs/iaTregs, that is antigenically driven by the inflammatory synovium environment. The dynamic interplay between CPLs/iaTregs, could be titled towards suppression evasion through IFNg, allowing for continual synovium inflammation. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.